Graeme E. Price

TLR3-Specific Double-Stranded RNA Oligonucleotide Adjuvants Induce Dendritic Cell Cross-Presentation, CTL Responses, and Antiviral Protection

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α+ and CD103+ DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1+ endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-β, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8+ T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses.

Immunization against conserved virus components induces broad, heterosubtypic protection against diverse influenza A viruses, providing a strategy for controlling unexpected outbreaks or pandemics until strain-matched vaccines become available. This study characterized immunization to nucleoprotein (NP) and matrix 2 (M2) by DNA priming followed by parenteral or mucosal boosting in mice and ferrets. DNA vaccination followed by boosting with antigen-matched recombinant adenovirus (rAd) or cold-adapted (ca) influenza virus provided robust protection against virulent H1N1 and H5N1 challenges. Compared to other boosts, mucosal rAd induced stronger IgA responses, more virus-specific activated T-cells in the lung, and better protection against morbidity following challenge even eight months post-boost. In ferrets, both mucosal and parenteral rAd boosting protected from lethal H5N1 challenge. These findings demonstrate potent protection by vaccination highly focused on conserved antigens and identify immune response measures in mice that differed among vaccinations and correlated with outcome.

Single-Dose Mucosal Immunization with a Candidate Universal Influenza Vaccine Provides Rapid Protection from Virulent H5N1, H3N2 and H1N1 Viruses

Background The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein. Methodology/Principal Findings In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals. Conclusion/Significance Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.

Cross-protective immunity to influenza A viruses

Antigenic changes in influenza virus occur gradually, owing to mutations (antigenic drift), and abruptly, owing to reassortment among subtypes (antigenic shift). Availability of strain-matched vaccines often lags behind these changes, resulting in a shortfall in public health. In animal models, cross-protection by vaccines based on conserved antigens does not completely prevent infection, but greatly reduces morbidity, mortality, virus replication and, thus, viral shedding and spread. Such immunity is especially effective and long-lasting with mucosal administration. Cross-protective immunity in humans is controversial, but is suggested by some epidemiological findings. ‘Universal’ vaccines protective against all influenza A viruses might substantially reduce severity of infection and limit spread of disease during outbreaks. These vaccines could be used ‘off the shelf’ early in an outbreak or pandemic, before strain-matched vaccines are available.

Comparison of vaccines for induction of heterosubtypic immunity to influenza A virus: Cold-adapted vaccine versus DNA prime-adenovirus boost strategies

Influenza epidemics or pandemics can arise for which strain- or subtype-matched vaccines are unavailable. Heterosubtypic immunity (Het-I) targeting conserved influenza A antigens could reduce morbidity and mortality during preparation of matched vaccines. Various vaccines inducing Het-I in animals have been studied separately using different viruses and conditions, but effectiveness for inducing Het-I has not been directly compared. The present studies compared immunization with cold-adapted (ca) viruses to DNA prime-recombinant adenovirus (rAd) boost vaccination to conserved antigens nucleoprotein (NP), matrix-2 (M2), or A/NP+M2. Both ca and DNA-rAd vaccinations induced antibody and T cell responses, and protected against lethal H1N1 challenge. Only A/NP+M2 DNA-rAd protected against challenge with highly pathogenic A/Vietnam/1203/2004 (H5N1); ca vaccine did not. Existing ca vaccines may provide some Het-I, but experimental vaccination focusing on conserved antigens was more effective in this model for protection against a divergent, highly pathogenic virus.

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

Background The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus. Methodology/Principal Findings BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus. Conclusion/Significance Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic.

Impact of cross-protective vaccines on epidemiological and evolutionary dynamics of influenza

Large-scale immunization has profoundly impacted control of many infectious diseases such as measles and smallpox because of the ability of vaccination campaigns to maintain long-term herd immunity and, hence, indirect protection of the unvaccinated. In the case of human influenza, such potential benefits of mass vaccination have so far proved elusive. The central difficulty is a considerable viral capacity for immune escape; new pandemic variants, as well as viral escape mutants in seasonal influenza, compromise the buildup of herd immunity from natural infection or deployment of current vaccines. Consequently, most current influenza vaccination programs focus mainly on protection of specific risk groups, rather than mass prophylactic protection. Here, we use epidemiological models to show that emerging vaccine technologies, aimed at broad-spectrum protection, could qualitatively alter this picture. We demonstrate that sustained immunization with such vaccines could—through potentially lowering transmission rates and improving herd immunity—significantly moderate both influenza pandemic and seasonal epidemics. More subtly, phylodynamic models indicate that widespread cross-protective immunization could slow the antigenic evolution of seasonal influenza; these effects have profound implications for a transition to mass vaccination strategies against human influenza, and for the management of antigenically variable viruses in general.

Mucosal Immunization with a Candidate Universal Influenza Vaccine Reduces Virus Transmission in a Mouse Model

ABSTRACT Pandemic influenza is a major public health concern, but conventional strain-matched vaccines are unavailable early in a pandemic. Candidate “universal” vaccines targeting the viral antigens nucleoprotein (NP) and matrix 2 (M2), which are conserved among all influenza A virus strains and subtypes, could be manufactured in advance for use at the onset of a pandemic. These vaccines do not prevent infection but can reduce disease severity, deaths, and virus titers in the respiratory tract. We hypothesized that such immunization may reduce virus transmission from vaccinated, infected animals. To investigate this hypothesis, we studied mouse models for direct-contact and airborne transmission of H1N1 and H3N2 influenza viruses. We established conditions under which virus transmission occurs and showed that transmission efficiency is determined in part at the level of host susceptibility to infection. Our findings indicate that virus transmission between mice has both airborne and direct-contact components. Finally, we demonstrated that immunization with recombinant adenovirus vectors expressing NP and M2 significantly reduced the transmission of virus to cohoused, unimmunized mice in comparison to controls. These findings have broad implications for the impact of conserved-antigen vaccines, not only in protecting the vaccinated individual but also in protecting others by limiting influenza virus transmission and potentially reducing the size of epidemics. IMPORTANCE Using a mouse model of influenza A virus transmission, we demonstrate that a candidate “universal” influenza vaccine both protects vaccinated animals from lethal infection and reduces the transmission of virus from vaccinated to nonvaccinated mice. This vaccine induces immunity against proteins conserved among all known influenza A virus strains and subtypes, so it could be used early in a pandemic before conventional strain-matched vaccines are available and could potentially reduce the spread of infection in the community.

Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector, PanAd3, Derived from the Bonobo Pan paniscus

Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BackgroundThe genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system.MethodsTo test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs.ResultsCross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced.ConclusionOur findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.